Deuteration of heptamethine cyanine dyes enhances their emission efficacy

The design of bright short-wave infrared fluorophores remains a grand challenge. Here we investigate the impact of deuteration on the properties in a series of heptamethine dyes, the absorption of which spans near-infrared and SWIR regions. We demonstrate that it is a generally applicable strategy that leads to enhanced quantum yields of fluorescence, longer-lived singlet excited states and suppressed rates of non-radiative deactivation processes.


Materials and Methods
Reagents and solvents of the highest purity available were used as purchased, or they were purified/dried using standard methods when necessary.The intermediates were synthesized according to the published procedures (10 1 , 11 2 , 12 3 , 13 4,5 ) or purchased (4H) from standard suppliers (Merck, TCI, Across Organics, etc.).Flash column chromatography was performed using silica gel (230−400 mesh). 1 H NMR spectra were recorded on 400 or 500 MHz spectrometers; 13 C NMR spectra were obtained on 125 MHz instruments in CDCl3, CD3OD, and d6-DMSO. 19F NMR were obtained on 376 MHz or 470 MHz instruments. 1 H chemical shifts are reported in ppm relative to CDCl3 (δ = 7.26 ppm), CD3OD (δ = 3.31 ppm) and d6-DMSO (δ = 2.50 ppm) as an internal reference. 13C chemical shifts are reported in ppm with CDCl3 (δ = 77.67 ppm), CD3OD (δ = 49.30ppm) and d6-DMSO (δ = 39.52 ppm) as internal references. 2 H NMR spectra were measured using nondeuterated solvents with addition of 5 μL of respective deuterated solvent to allow locking of a sample.Deuterated solvents were kept under nitrogen atmosphere.HRMS of the synthesized compounds was obtained using a triple quadrupole electrospray ionization mass spectrometer in a positive or negative mode coupled with direct inlet.The relative ratio of d5:d6:d7 for the final cyanines 1-4D was calculated from isotope pattern of the molecular peak detected by HRMS.

Steady-State Absorption and Emission Spectroscopy
Absorption spectra and molar absorption coefficients were obtained on a UV-vis spectrometer with matched 1.0 cm quartz cells in CH2Cl2 (1-3) or MeOH (4), with constant amount 0.4% of DMSO.Fluorescence spectra were measured using a fluorescence spectrometer in a 1.0 cm quartz fluorescence cuvette at 20 °C in CH2Cl2.The sample concentrations were adjusted to keep the absorbance below ~0.2 at the corresponding excitation wavelength.Each sample was measured five times, and the spectra were averaged.Emission spectra were normalized and corrected by the photomultiplier sensitivity function using correction files supplied by the manufacturer.
Quantum yields of fluorescence were measured in CH2Cl2 against the reference using the relative method.For compounds 1 and 2, IR-1061 was used as a reference standard (ΦF = 0.32% 6 in CH2Cl2).For compounds 3 and 4 dye 4H (ICG) was used as a reference standard (ΦF = 12% 7 in methanol), and the calculations were done using eq.bellow for correction of refractive indexes.
The subscripts S and R refer to the sample and reference, respectively.ΦR is the known quantum yield of the reference standard, slope is the slope of integrated fluorescence spectrum intensity plotted against absorbance A of the solution at the excitation wavelength (λex), and n is the refractive index of the solvent, using values nS = 1.4244 for CH2Cl2 and nR = 1.4793 for methanol.Each compound was measured using 15 independent solutions with absorbance in a range 0.02-0.2 at a respective excitation wavelength and compared to reference sample measured under the same settings using 10-15 independent solutions with absorbance in a range 0.02-0.2.Error calculation of the quantum yield was propagated from the error in slope of both the reference and the unknown.

Stability of 1 DD In the Dark and Under Irradiation
Compounds 1H and 1DD were dissolved in CH2Cl2 (A=1 at 988 nm) and irradiated with LEDs at 820 nm (~25 mW/cm 2 ) and the progress of the irradiation was monitored at periodic intervals (10 min for irradiated, 30 min for the experiment in the dark) by UV−vis spectrometry.The stability of 1H and 1DD in the dark was recorded using the same procedure with exclusion of the irradiation source.

Time-Resolved Emission Spectroscopy
Time-correlated single photon counting was employed for the time resolved emission measurement of 1-4D in solution (DCM).A 780 nm pulsed diode laser (PicoQuant LDH-P-C-780) with a repetition rate of 80 MHz was used as the excitation source.An epifluorescent configuration was used to collect emission with a 650 long pass dichroic mirror (Thorlabs DMLP650R).The laser was filtered using 830 nm longpass filter (Newport Optics 10CGA-830).The emission was fiber coupled into reflective collimators(Thorlabs RC12FC-P01) and detected using superconducting nanowire single-photon detectors (Quantum Opus Opus One).
A timing module recorded all photon events (Picoquant HydraHarp 400) in time-tagged timeresolved mode.
Given the short lifetimes of these dye, lifetimes were fit using a single exponential with an offset from the peak maximum based on the Gaussian instrument response function (46 ps for all but 1DD and 17 ps for 1DD) (Figure S1).A overlay of 4H and 4D is provided to show the difference in lifetimes (Figure S2).

General Procedure for the Synthesis of 3H-D and 4D.
The Schiff base 6H or 6D (1 eq., 0.33 mmol) was dissolved in dry MeCN (5 mL) and kept under N2 atmosphere.DIPEA (3 eq., 127 mg, 0.98 mmol) was added dropwise, and the mixture was cooled in ice bath, followed by the addition of Ac2O (5 e.q., 0.15 mL, 1.6 mmol).The red solution turned yellow within few minutes.Heterocycle 11 or 12 (3 eq., 0.98 mmol) was then dissolved in MeCN/CH3OH or MeCN-d3/CD3OD (1.5 ml; 2:1) with DIPEA (3 eq., 127 mg, 0.98 mmol) and the solution was added to the reaction mixture.The reaction flask was covered in aluminum foil and stirred at room temperature for 16 h.The volatiles were evaporated under reduced pressure to give the crude product, to which Et2O (5 mL) was added, the precipitate was filtered off and washed with water (5×10 mL) and diethyl ether (3×10 mL) and dried under reduced pressure to give the target cyanines 3H-4D if not stated differently.

HRMS Spectroscopy
Calculation the Relative ratio of d5:d6:d7 Cyanines The relative ratio of d5:d6:d7 for the final cyanines 1-4D was calculated from isotope pattern of the molecular peak detected by HRMS.Based on the relative abundance of the respective masses compared to the expected isotopic pattern ratios for each of the derivatives d5 -d7 the relative ratio was calculated.The d5 isomers were taken as a referential starting point due to their specific main peak (100% abundance) not coinciding with other isotopic values.Using 1H-D as an example, the intensity of mass corresponding to d5 experiences contribution by the d5-isomer: [d5] = I(M), where M corresponds to the calculated exact mass of d5-isomer The mass corresponding to d6-1D experiences contribution from d6-1D and the isotope pattern of d5-1D, which can be calculated from the molecular formula (e.g. in ChemDraw): [d6] + [d5] × 0.487 = I(M+1), where M+1 corresponds to the calculated exact mass of the d6isomer.
The mass corresponding to d7-1D experiences contribution from d7-1D and the isotope patterns of d6-and d5-1D, which can be calculated from the molecular formula (e.g. in ChemDraw):

Figure S2 :
Figure S2: Comparison of the TCSPC lifetime trace of dye 4H and 4D to show that deuteration increases lifetime.

Figure S47 .
Figure S47.Dependence of absorption at λmax on the concentration of 1H (left) and 1D (right) in DCM.

Figure S48 .
Figure S48.Dependence of absorption at λmax on the concentration of 1DD in DCM.

Figure S49 .
Figure S49.Dependence of absorption at λmax on the concentration of 2H (left) and 2D (right) in DCM.

Figure S50 .
Figure S50.Dependence of absorption at λmax on the concentration of 3H (left) and 3D (right) in DCM.

Figure S51 .
Figure S51.Dependence of absorption at λmax on the concentration of 4H (left) and 4D (right) in MeOH.

Figure S52 .
Figure S52.Dependence of the integral of emission on the absorption at λexc nm for 1H (left) and 1D (right) in DCM.

Figure S53 .
Figure S53.Dependence of the integral of emission on the absorption at λexc nm for 1H in DCM (depicted in red) and DMSO (depicted in black).

Figure S56 .
Figure S56.Dependence of the integral of emission on the absorption at λexc nm for 3H (left) and 3D (right) in DCM.

Figure S57 .
Figure S57.Dependence of absorption at λ= 750 nm on the area of emission of 4H (left) and 4D (right) in DCM.

Figure S59 .
Figure S59.(left) The F data for 1H-DD from Figure S51 and S53 plotted in a single graph.(right) The F data for 4H-D from Figure S56 plotted in a single graph.

Figure S60 .
Figure S60.Enhancement () of F in 1D-4D as a function of the HOMO-LUMO gap.

Table S3 .
Statistical analysis of the relative F values.Comparison of stability of 1H (red) and 1DD (black) in DCM in the dark (dotted line) and under irradiation with light at 820 nm.